Enhancing the efficacy of immunotherapies by supplementing with complement

ABSTRACT

The present invention is directed to a composition and method for enhancing the complement-mediated cytotoxicity of therapeutic antibodies for their target cells (i.e. those cells displaying the target epitope). More particularly the present invention enhances the efficacy of such therapies by providing a supplemental source of complement

RELATED APPLICATIONS

[0001] This application claims priority under 35 USC §199(e) to U.S.Provisional Application Ser. Nos. 60/311,451, filed Aug. 10, 2001,60/318,295, filed Sep. 10, 2001, 60/373,785, filed Apr. 19, 2002 and60/374,336, filed Apr. 22, 2002, the disclosures of which areincorporated herein.

FIELD OF THE INVENTION

[0002] The present invention is directed to novel therapeuticcompositions and an improved method of treating illness throughimmunotherapy. The improvement relates to providing a supplementalsource of complement to enhance the cytotoxic activity of therapeuticantibodies for those cells that display the target epitope.

BACKGROUND OF THE INVENTION

[0003] There is a voluminous literature which describes the use of mAbsin the treatment of cancer, including antibodies against B-cellmalignancies as described in U.S. Pat. Nos. 4,987,084 and 6,183,744, thedisclosures of which are incorporated herein. These anti-cancer mAbsfunction by binding specifically to a tumor or circulating cancer cell,and after binding the mAb, killing the cancer cell by one or moremechanisms. These mechanisms can include complement-mediatedcytotoxicity (CMC) or opsonization; antibody-dependent cell mediatedcytotoxicity (ADCC); induction of programmed cell death (Apoptosis);interference with a particular cell function by blocking a receptor orupregulating/downregulating a particular signaling process. Many mAbbased therapies have failed because the mAbs did not facilitate one ormore of these functions efficiently.

[0004] With respect to complement activation, it is now well-recognizedthat one of the mechanisms by which cancer cells evadecomplement-mediated lysis or opsonization is by upregulating normalcomplement control proteins. In particular, CD46, CD55, and CD59 may beexpressed at increased levels on cancer cells (compared to normal cells)and thus they are not killed by complement activation. However, there isyet another reason why a mAb may not be able to induce ADCC oropsonization of a tumor or cancer cell. Quite often complement levelsare substantially reduced in cancer patients due to their disease. Moreparticularly, one or more components of the complement cascade may besufficiently reduced so that the mechanisms by which the mAb usescomplement to facilitate cell killing (opsonization or direct lysis(CMC)) may not work effectively.

[0005] Currently there are several mAbs that are being used to treatcancer including Rituximab (Idec, chimeric), B1(Coulter mouse IgG2a),Panorex (Glaxo IgG2a), C225 (Imclone chimeric IgG), Vitaxin (Medimmunechimeric IgG), Smart M195 and 1D10 (PDL humanized IgG mAbs) and Campath(Berlex humanized IgG1). In particular, one of these mAbs, Rituximab,has been FDA-approved for the treatment of Non-Hodgkin's Lymphoma. Themechanism used by these antibodies to effect cancer cell death iscurrently subject to debate. Specifically, a number of investigatorscontend that Rituximab kills via non-complement mechanisms includingApoptosis, and Fc receptor mediated phagocytosis. However, the presentinvention is based in part on applicants' belief that both Rituximab andCampath mediate cell killing predominantly through complementactivation. Since the role of complement in the mechanism of action ofRituximab is still being debated, the importance of measuring complementand supplementing its levels in patients being administered therapeuticantibodies has not been previously considered. Applicants' own work (seeExample 1) strongly suggests that the only important mechanism by whichRituximab mediates killing of cancer cells is through complement.

[0006] It has been reported that treatment of patients with Rituximabfor Non-Hodgkin's Lymphoma and other B Cell lymphomas typically gives aresponse rate of 50%. Experiments conducted in applicants' laboratoryindicate that the active form of Rituximab (RTX) in vivo is RTXcovalently associated with C3b(i). It is believed that C3b(i) bound toRTX or other therapeutic mAbs provides a platform targeting epitope. IfRituximab's mechanism of action requires robust complement activation,then the reduced levels of complement proteins that are often observedin cancer patients may be the cause of many of the patients' failure torespond to Rituximab therapy. Furthermore, even if a patient initiallyhas an adequate supply of complement, if that patient's tumor burden issubstantial, treating with large amounts of a mAb whose mechanism ofaction requires complement activation may result in the availablecomplement being used up or substantially depleted. Moreover, megadosingwith additional mAb will not resolve the underlying problem relating tothe lack of available complement and will not increase the effectivenessof the treatment.

[0007] The effectiveness of RTX, and other therapeutic mAbs, can beenhanced, however, if complement is restored with fresh plasma, or withindividual components of the complement system (either purified naturalcomponents or recombinantly produced complement components). Theimprovement relates to identifying patients that will be refractory foranti-cancer passive immunotherapy due to inadequate levels of complementactivity, and enhancing the efficacy of such therapy by providing asupplemental source of complement proteins.

SUMMARY OF THE INVENTION

[0008] The present invention is directed to compositions and methods forenhancing the complement-mediated cytotoxic efficacy of antibody basedanti-tumor therapies. In particular, the present invention is directedto enhancing the efficacy of Rituximab and other anti-tumor mAbtreatments by providing patients with a supplemental source ofcomplement, either as fresh plasma/sera or as isolated complementcomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1A & 1B are bar graphs representing the results of anexperiment in which flow cytometry was used to measure RTX-mediatedkilling of DB cells over the course of a 24 hour incubation. At cellconcentrations of 3.6×10⁶, use of 100 ug/ml RTX leaves more than half ofthe cells alive after 24 hours. Supplementation of the serum with C2leads to a substantial increase in killing, as illustrated in the largedrop in live cells in the presence of both RTX and C2. Note that C2 byitself in serum does not promote killing.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Definitions

[0011] In describing and claiming the invention, the followingterminology will be used in accordance with the definitions set forthbelow.

[0012] The following abbreviations are used throughout the applicationand have the following meanings: C=complement; RTX=Rituximab; NHS=NormalHuman Serum (a normal complement source); CP=citrated human plasma (alsoa complement source).

[0013] As used herein, the term “nucleic acid” encompasses RNA as wellas single and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention.

[0014] The term “peptide” encompasses a sequence of 3 or more aminoacids wherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

[0015] 1. peptides wherein one or more of the peptidyl —C(O)NR— linkages(bonds) have been replaced by a non-peptidyl linkage such as a —CH²⁻carbamate linkage (—CH₂OC(O)NR—), a phosphonate linkage, a —CH²⁻sulfonamide (—CH_(2—)S(O)₂NR—) linkage, a urea (—NHC(O)NH—) linkage,a—CH₂— secondary amine linkage, or with an alkylated peptidyl linkage(—C(O)NR—) wherein R is C₁-C₄ alkyl;

[0016] 2. peptides wherein the N-terminus is derivatized to a —NRR₁group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)₂R group,to a —NHC(O)NHR group where R and R₁ are hydrogen or C₁-C₄ alkyl withthe proviso that R and R₁ are not both hydrogen;

[0017] 3. peptides wherein the C terminus is derivatized to —C(O)R₂where R₂ is selected from the group consisting of C₁-C₄ alkoxy, and—NR₃R₄ where R₃ and R₄ are independently selected from the groupconsisting of hydrogen and C₁-C₄ alkyl.

[0018] Naturally occurring amino acid residues in peptides areabbreviated as recommended by the IUPAC-IUB Biochemical NomenclatureCommission as follows: Phenylalanine is Phe or F; Leucine is Leu or L;Isoleucine is Ile or I; Methionine is Met or M; Norleucine is Nle;Valine is Vat or V; Serine is Ser or S; Proline is Pro or P; Threonineis Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is Hisor H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K;Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys orC; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G,and X is any amino acid. Other naturally occurring amino acids include,by way of example, 4-hydroxyproline, 5-hydroxylysine, and the like.

[0019] As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially free(i.e. at least 60% free, preferably 80% free, and most preferablygreater than 90% free) from other components with which they arenaturally associated.

[0020] As used herein, the term “treating” includes alleviating thesymptoms associated with a specific disorder or condition and/orpreventing or eliminating said symptoms. For example, treating cancerincludes preventing or slowing the growth and/or division of cancercells as well as killing cancer cells.

[0021] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

[0022] As used herein, the term “parenteral” includes administrationintraperitoneally, intraarterially, subcutaneously, intravenously orintramuscularly.

[0023] As used herein a “substantially reduction in complement activity”is considered to be any reduction in activity that is greater than 25%.

[0024] The Invention

[0025] The present invention is based on the observation that antibodybased anti-cancer therapies such as Rituximab therapy utilize amechanism of action that requires complement activation. Quite oftencomplement levels are substantially reduced in cancer patients due totheir disease. More particularly, one or more components of thecomplement cascade may be sufficiently reduced so that the mechanisms bywhich the mAb uses complement to facilitate cell killing (opsonizationor direct lysis (CMC) or killing promoted by complement receptors oneffector cells) may not work effectively. Furthermore, even in patientsthat initially display adequate levels of complement, those levels canbe substantially depleted over the course of treatment such thatinadequate levels of complement remain during the mid to later stages oftreatment. One aspect of the present invention is directed to a methodof monitoring the complement capabilities of patients to identifypatients that have substantially depleted complement activity. Anotherembodiment of the invention is directed to a method of enhancing theefficacy of an antibody based therapy by supplementing the treatmentwith either matched donor plasma or serum (as a source of complement) orpurified native or recombinant complement proteins.

[0026] If complement activation is required to facilitate a mAb-basedtherapy, then as a first step, the endogenous level of complementactivity in the patient's serum or citrated plasma should be measured.In accordance with one embodiment a method is provided for screeningpatients to identify individuals that will be refractory to antibodybased anti-cancer therapies such as Rituximab therapy. Moreparticularly, one aspect of the present invention is directed to amethod of determining a patient's capacity for complement action as anindication of their responsiveness to passive immunotherapy. The methodcomprises the step of measuring the complement capacity (i.e. the actuallevels of complement proteins and complement activity) of theindividual. These measurements may include CH50 titrations as well asmeasurement of the levels of individual complement proteins, todetermine if one or more of them are lacking. CH50 assays have beenpreviously described and are well known to those skilled in the art (seeFetterhoff et al., (1984) J. Clin. Lab Immunol 14: 205).

[0027] In one embodiment the patient's complement capacity is determinedby measuring the ability of the patient's serum/plasma to promotecomplement-mediated opsonization or killing of cancer cells uponaddition of adequate amounts of the anti-cancer antibody. The cancercells used in such an assay can be isolated from the patient to betreated (e.g. a biopsy sample or isolated from the bloodstream) or takenfrom established cell lines that serve as model systems for theparticular cancer type afflicting the patient. Opsonization includesdeposition of activated fragments of component proteins C3 and/or C4 onthe cancer cells. Alternatively, the patient's complement capacity maybe determined by measuring the patient's CH50. Those individuala thathave substantially reduced complement capacity will be refractory forantibody based therapies that require robust complement activity, suchas Rituximab therapy.

[0028] In one embodiment, the measurement of a patient's complementcapacity is made using the patient's serum or citrated plasma (both areacceptable complement sources) and compared to the measurement obtainedusing compatible donor-matched serum or citrated plasma that is known tohave a normal and robust level of complement activity. If the patient'scomplement levels are deemed adequate, then the mAb based therapy can beinitiated. However, the complement capacity of the patient still needsto be monitored throughout the treatment to ensure adequate levels ofcomplement are maintained during the entire treatment.

[0029] One aspect of the present invention relates to a method ofimproving the effectiveness of antibody mediated anti-cancer therapies.The method comprises the step of measuring complement levels in apatient prior to administering a dose of the therapeutic anti-neoplasticantibody. The actual levels of complement proteins and complementactivities are often reduced in patients suffering from a variety ofdifferent cancers. In addition, the patient's ability to replacecomplement proteins may be impaired or the tumor burden may be so greatthat sufficient complement is not present for the entire treatment. Whena patient's capacity for complement activation is low, as determined forexample, by a low CH50 level, then the administration of the anti-cancerantibody therapy can be supplemented with fresh plasma/serum from acompatible donor or with purified components from the complementcascade. Typical CH50 levels in healthy individuals with functionalcomplement will range from about 100 to about 300. Any individual thathas a CH50 level of 150 or lower will likely receive benefit fromadministration of exogenous, supplemental complement proteins.

[0030] Alternatively, measurements of complement activity, as defined byassays that combine an anti-cancer antibody (such as Rituximab), cancercells and the patients serum/plasma, may be required to identityindividuals likely to benefit from treatment. Reduction of efficacy by afactor of two or more in either opsonization or cell killing willprovide an approximate standard for such identification. Furthermore,the assay can be extended to confirm, or identify, those patients thatwould benefit from supplementing immunotherapy with a source ofcomplement proteins. The assay comprises obtaining a serum or plasmasample from the patient and measuring the ability of the patient'sserum/plasma to promote complement-mediated opsonization or killing ofcancer cells upon addition of adequate amounts of the anti-cancer cancerantibody in the presence and absence of complement. When significantadditional opsonization or killing of cancer cells occurs in thepresence of supplemental complement, then administration of supplementalcomplement in conjunction with the anti-cancer antibody therapy iswarranted. The cancer cells used in such an assay can be isolated fromthe patient to be treated (e.g. a biopsy sample or isolated from thebloodstream) or taken from established cells lines that serve as modelsystems for the particular cancer type afflicting the patient.Opsonization includes deposition of activated fragments of componentproteins C3 and/or C4 on the cancer cells.

[0031] In accordance with one embodiment a method of identifyingindividuals who would benefit from complement supplementation of ananti-cancer immunotherapy is provided. The method comprises the steps ofobtaining a serum or plasma sample from the patient to be treated andcontacting the serum or plasma sample with cancer cells and theanti-cancer antibody to form a reaction mixture. The reaction mixture isthen incubated for a predetermined length of time in the presence andabsence of donor complement proteins. A determination is then made ofwhether the serum or plasma sample exhibits higher opsonization or cellkilling in the presence of the supplemental donor complement proteins.Those patients whose serum/plasma give a higher level of opsonization orcell killing in the presence of supplement complement are thusidentified as individuals whose anti-cancer antibody therapy wouldbenefit from complement supplementation.

[0032] In this method, the serum or plasma sample is typically isolatedfrom the patient, combined with the relevant cancer cells (i.e. cancercells isolated from the patient or an established cell line that servesas a model for the patient's cancer) and the therapeutic anti-cancerantibody, and then divided into two separate reaction mixtures.Supplemental donor complement proteins are then added to the firstreaction mixture and not the second reaction mixture. Enhancedopsonization or cell killing that occurs in the first reaction mixturerelative to the second reaction mixture indicates that supplementationwith donor complement would be beneficial in enhancing the in vivoefficacy of the anti-cancer immunotherapy.

[0033] Donor serum or citrated plasma is used in one embodiment toprovide a source of complement, however individual complement componentscan also be used to supplement patient complement activity. Inaccordance with one embodiment an improved composition is provided fortreating cancer patients with an antibody based anti-cancer therapy,wherein the patient exhibits substantially lower levels of complementactivity before or during the treatment. A detected substantialreduction in complement activity (i.e. greater than 25%) resulting fromantibody based anti-cancer cancer therapy will lead to complementsupplementation in accordance with the present invention. Moreparticularly, cancer patients having CH50 levels, at any time, of lessthan 150, or more preferably less than 100, would benefit from theadministration of the improved immunotherapy compositions of the presentinvention. The improved composition comprises an anti-neoplasticantibody and a composition comprising a complement protein. Inaccordance with one embodiment the complement protein is provided as afresh serum or fresh citrated plasma isolated from a compatible donor.In one embodiment the serum/plasma is prepared from individuals havingAB blood type.

[0034] The amount of donor serum or plasma that will be administered toan individual will be varied base on the severity of complementdeficiency in the patient. However, typically the patient will beadministered about one to two units of plasma or serum The donorserum/plasma can be administered separately or as an admixture withanti-cancer antibody.

[0035] In addition to, or as an alternative to, using serum or plasma tosupplement patient complement function during anti-cancer immunotherapy(or other antibody-based therapy that relies on complement fixation forits mechanism of action), individual purified complementproteins/factors can be used. The purified complement proteins/factorscan be isolated from natural sources or more preferably the proteins arerecombinantly produced and purified. In one embodiment the purifiedcomplement proteins/factors are selected from the group consisting ofC1-C9, factor B, factor D and properdin and more preferably selectedfrom the group consisting of C1, C2, C3, C4 and C5. In one embodimentthe purified complement proteins/factors are selected from the groupconsisting of C1, C2, and C5. Accordingly, an improved immunotherapycomposition is provided wherein the composition comprises one or moreanti-tumor antibodies in combination with one or more complementproteins/factors.

[0036] Preferably, the complement factors used in conjunction with theanti-cancer antibody therapy will be selected based on their prevalencein the patient to be treated. In particular, it is known that certaincomponents of the complement cascade are more prevalent in humans andother mammals than other components. Since complement protein C2 istypically low relative to the other complement proteins, one aspect ofthe present invention comprises enhancing the effectiveness ofanti-cancer antibody therapy (such as RTX treatment) by supplementationwith purified human complement protein C2. In addition it has beenreported that the oxidized form of C2 has been shown to be aparticularly effective form of that complement protein in facilitatingand prolonging complement activation. Accordingly, one embodiment of thepresent invention encompasses supplementing an antibody therapy (thatrelies on complement fixation for its mechanism of action) with theadministration of oxidized C2.

[0037] Preferably the compositions of the present invention furthercomprises a pharmaceutically acceptable carrier. A pharmaceuticalcomposition comprising one or more individual complement proteins(recombinantly produced or purified natural components, or matched donorserum/plasma) and a pharmaceutically acceptable carrier can beadministered separately or as an admixture with the anti-cancerantibody. The composition can be administered to the target cells usingany of the known routes including oral, parenteral, transdermal or timerelease implant. In one preferred embodiment the composition isadministered intravenously.

[0038] In addition to RIX, other mAbs have been described and are underinvestigation for the immunotherapy of cancer, including: B1, Coultermouse IgG2a; Panorex, Glaxo IgG2a; C225, Imclone chimeric IgG; Vitaxin,Medimmune chimeric IgG; Campath, Berlex humanized IgG1; Smart M195 and1D10, PDL humanized IgG mAbs; OvaRex, AltaRex Corp. murine antibody forovarian cancer; BEC2, ImClone Systems Inc., murine IgG for treatment oflung cancer; IMC-C225, ImClone Systems Inc., chimeric IgG for treatmentof head and neck cancer; Vitaxin, MedImmune humanized antibody fortreatment of sarcoma; LymphoCide, Immunomedics humanized IgG fortreatment of non-Hodgkin's lymphoma; Oncolym, Techniclone, Inc., murineantibody for treatment of non-Hodgkin's lymphoma; use of the anti-C3b(i)mAb 3E7 (or the next generation mAb) in a radioactive form: 131-I, or90-Y; as well as Alemtuzumab; Ibritumomab; Gemtuzumab; Epratuzumab;Apolizumab; HuM195; Trastuzumab; Cetuximab; Edrecolomab; ABX-EGF; 2C4;DM-1; ING-1; Bevacizumab; and Anti-KDR antibodies. Any of theseantibodies whose mechanism of action is primarily through complement arecandidates for supplementation with complement to enhance the cytotoxicactivity of the antibody for its target cells (i.e. those cellsdisplaying an epitope that the antibody specifically binds).Accordingly, it is anticipated that the effectiveness of each of theseantibodies could be enhanced if the therapy includes supplementationwith an exogenous source of complement (either from matched donorserum/plasma or from supplementation with individual complementproteins).

[0039] In accordance with one embodiment of the present invention amethod of enhancing the cytotoxic activity of anti-neoplastic antibodiesfor their target cells is provided. The method comprises the steps ofcontacting the target cells with one or more anti-neoplastic antibodiesand contacting the target cells with a composition comprising donorhuman complement proteins. The target cells can be contacted either invitro or in vivo with the anti-neoplastic antibodies and the donorcomplement proteins, in any particular order or the two can beadministered simultaneously. In one preferred embodiment the targetcells are first contacted with the donor complement and then theanti-neoplastic antibody is added for contact with the target cells.When the target cells are contacted in vivo the compositions comprisingthe anti-neoplastic antibody and the complement are typicallyadministered intravenously, although other parenteral routes ofadministration are also possible.

[0040] In one embodiment the donor complement composition comprisesfresh serum or citrated plasma, prepared from a human donor having amatched blood type and optionally supplemented with purifiedrecombinantly produced complement proteins such as C2. In one embodimentthe human donor for the plasma/serum has AB blood type. Alternatively,the donor complement composition comprises one or more purified membersof the complement cascade, including proteins produced using recombinanttechnologies. In one preferred embodiment the composition comprisescomplement protein C2, in particular, oxidized C2.

[0041] In one embodiment a method is provided for treating a B-cellmalignancy. The method comprises administering to a human subject havinga B-cell malignancy a therapeutic composition comprising apharmaceutically acceptable carrier and an anticancer antibody specificfor B-cells as well as a composition comprising a source of complementfactors. The two compositions can be administered simultaneously orseparately and in one embodiment the two are combined to form a singlecomposition comprising an anticancer antibody specific for B-cells, asource of complement factors and a pharmaceutically acceptable carrier.In one embodiment the composition is administered parenterally(preferably intravenously) either continuously or in multiple doses. Inone embodiment the anticancer antibody component comprises at least twomonoclonal antibodies that bind with distinct epitopes uniquelyexpressed on cancer cells. This method can be used to treat variousB-cell malignancies including indolent forms of B-cell lymphomas,aggressive forms of B-cell lymphomas, chronic lymphocytic leukemias, andacute lymphocytic leukemias, and in one embodiment the method is used totreat non-Hodgin's lymphoma. The therapeutic composition can furtherinclude additional anticancer agents that are known to those skilled inthe art, including cytokines and chemotherapeutic agents.

[0042] The method of enhancing an antibody's effectiveness in killingits target cells by supplementing with exogenous complement can be usedwith any mAb-based therapy whose action is believed to requirecomplement activation. Such therapies can include cancer as well asother diseases including infectious disease or conditions in whichcertain cells are targeted to increase or decrease a particularbiological activity. The patient can be monitored before and during thecourse of treatment with a therapeutic antibody. When the patient'scomplement drops below a certain threshold (for example a CH50 of lessthan 120), then the patient is administered an intravenous infusion withcompatible serum/plasma from a normal individual with sufficiently highlevels of complement activity (or an effective amount of one or morerecombinantly produced complement proteins) either before orsimultaneously with subsequent doses of the therapeutic antibody.

[0043] Accordingly, the present invention is also directed to the use ofsupplemental complement (comprising either donor serum/plasma orindividual complement proteins such as C2) to enhance the effectivenessof antibody therapy for any antibody-based treatment that involves adisease wherein complement is low. For example, diseases other thancancer that often have low complement titers include diseases that arecharacterized by inflammation, such as infectious disease, autoimmunedisorders (e.g. lupus erythematosus and rheumatoid arthritis) orconditions in which certain cells are targeted for destruction orremoval as a means to increase or decrease a particular biologicalactivity. Autoimmune diseases can be treated with anti-B cellantibodies, such as Rituximab and such treatments can be supplementedwith complement factors in accordance with the present invention throughthe administration of normal compatible donor plasma.

[0044] Accordingly, the method of supplementing with complement can beused as a general strategy for enhancing the efficacy of anantibody-based therapy whose action is believed to require complementactivation, and where the afflicted individual has a lowered capacityfor complement activation. The method comprises the steps ofadministering complement factors that are low or missing from theindividual in conjunction with the administration of the mAb-basedtherapy. The supplemental complement factors can be administered eitherbefore or after the administration of the mAb-based therapy orsimultaneously with the therapeutic antibodies. In one preferredembodiment the supplemental complement factors are combined with thetherapeutic antibodies and administered as a single composition.

[0045] The present invention also encompasses a pack or kit comprising asource of complement for use with immunotherapy applications. Inaccordance with one embodiment a kit for enhancing the cytotoxicactivity of anti-tumor antibodies is provided wherein the kit compriseshuman serum or plasma (isolated from either an AB blood type donor or amatched blood type donor) or one or more purified complement proteins.The purified complement is preferably a recombinant produced proteinselected from the group consisting of C1, C2, C3, C4, C5, C6, C7, C8,C9, factor B, factor D and properdin. The genes encoding humancomplement proteins have been cloned and any reference herein to“recombinant complement proteins” is intended to cover recombinantlyproduced complement proteins that have the natural amino acid sequenceas well as proteins whose amino acid sequence has been modified (byamino acid deletions, additions or substitutions or by othermodifications) but still retain the native complement protein'sfunction. For example, the amino acid sequence of a complement proteinmay be modified to enhance its functional half life in vivo or enhanceits shelf life. In one embodiment the recombinant protein is selectedfrom the group consisting of C2, C3 and C4, with C2, and moreparticularly, oxidized C2 being the most preferred complement protein.In one embodiment the kit further comprises a therapeutic antibody suchas an anti-cancer antibody, including anti-cancer antibodies selectedfrom the group consisting of Rituximab and Campath.

[0046] The kits of the present invention may further comprise reagentsfor detecting and monitoring complement function in an individual. Moreparticularly the kit may include reagents for determining CH50 levelsand/or reagents for monitoring the opsonization of the target cells.Accordingly, the kit may include antibodies that are directed againstC3b(i), such as mAb 3E7 or 7C12. These antibodies may be derivatizedwith fluorescein or other fluorescent signaling moieties. The complementproteins and the therapeutic and diagnostic antibodies of the kit can bepackaged in a variety of containers, e.g., vials, tubes, microtiter wellplates, bottles, and the like. Other reagents can be included inseparate containers and provided with the kit; e.g., positive controlsamples, negative control samples, buffers, cell culture media, etc.Preferably, the kits will also include instructions for use.

EXAMPLE 1

[0047] Rituximab Mediates Cell Killing Through Complement Activation

[0048] To determine if complement activation is correlated with RTXbinding to cells, two CD20-positive cell lines were reacted with RTX ina variety of sera. The readout for complement activation in theseexperiments is the covalent deposition of activated fragments ofcomplement protein C3 (defined herein as C3b(i)) on the cells. Mab 3E7,which binds to activated C3b(i) bound to cells, was also used in theseexperiments to enhance RTX-mediated complement activation. Direct andindirect RIA was used to quantitate deposition of C3b(i) and binding ofmAb 3E7 to Raji or ARH-77 cells when a variety of NHS were used for Copsonization. Radiolabeled mAb 7C12, which binds to C3b(i), was used forthe primary readout.

[0049] The results in 50% NHS, summarized in Table 1, indicate that inthe presence of both RTX and mAb 3E7, >1 million molecules of C3b(i)bind per Raji cell, and reduced but still substantial C opsonizationoccurs in the presence of either one of the mAbs alone. Binding of RTXalone, in NHS, to both cell lines led to significant increases in C3b(i)opsonization. In the presence of RTX, mAb 3E7 appears to facilitate achain reaction in which binding of C3b(i) to the cells leads to bindingof mAb 3E7, which may either modestly activate C (mAb 3E7 is murineisotype IgG1) and/or enhance capture of additional molecules of C3b(i).RIA with ¹²⁵I-RTX revealed that 220,000±43,000 molecules (n=7) bound toRaji cells, and 310,000±60,000 molecules (n=4) bound to ARH-77 cells, inthe presence and absence of C. Approximately 1,000,000 molecules of mAb3E7 were found to bind to either of the cells in the presence of RTX andC (Table 1). These experiments thus also demonstrate amplification inthe sense that more molecules of C3b(i) are deposited on the cells thanbound RTX molecules. TABLE 1 C3b(i) and mAb 3E7 Bind to CD20⁺ Cellsduring C/RTX Opsonization Molecules Bound per Cell^(a) C3b(i)^(b)Anti-C3b(i) mAb 3E7^(c) Raji Cells incubated in: NHS   300,000 ±150,000^(d) (18) NA NHS/RTX   610,000 ± 190,000 (18) NA NHS/RTX/mAb1,400,000 ± 500,000 (13)   890,000 ± 220,000 (6) 3E7 NHS/mAb 3E7  680,000 ± 340,000 (14)   420,000 ± 190,000 (6) ARH-77 Cells incubatedin: NHS   150,000 ± 60,000 (10) NA NHS/RTX   790,000 ± 230,000 (10) NANHS/RTX/mAb   930,000 ± 240,000 (9) 1,100,000 ± 200,000 3E7 (3) NHS/mAb3E7   370,000 ± 110,000 (9)   240,000 ± 60,000 (3)

[0050] In summary, for Raji cells, either mAb singly or the combinationgave more C3b(i) deposition than NHS alone (p<10⁻³). RTX/mAb 3E7 gavemore C3b(i) deposition than either mAb alone (p<10⁻³). More mAb 3E7 wasbound in the presence of RTX (p=3×10⁻³). For ARH-77 cells, either mAbsingly or the combination gave more C3b(i) deposition than NHS alone(p<10⁻³). The combination gave more C3b(i) deposition than mAb 3E7 alone(p<10⁻³). More mAb 3E7 was bound in the presence of RTX (p=2×10⁻³).

[0051] Co-localization of RTX and mAb 3E7.

[0052] Fluorescence microscopy was used to address critical questionsraised by the RIA experiments concerning the molecular sites of bindingof C3b(i) and mAb 3E7 to RTX-opsonized CD20+ cells in the presence of C.When Raji or ARH-77 cells were incubated with red Alexa 594 RTX andgreen Alexa 488 mAb 3E7 (specific for C3b(i)) together in NHS orcitrated plasma, the two mAbs co-localize on the cells, as revealed bycoincidence of red and green fluorescence (identified with differentmicroscope filters). In the presence of mAb 3E7 cross-linking of thecells was often observed, as well as capping and/or co-localization ofthe probes to discrete areas on the cells. If cells are opsonized withred Alexa 594 RTX alone in citrated plasma, washed and then probed withgreen Alexa 488 mAb 3E7, this green mAb again co-localizes withpreviously bound red RTX, and in many cases cells are extensivelycross-linked. Binding of red RTX alone to cells in the presence orabsence of NHS leads to a more homogenous binding pattern and neithercapping nor cross-linking is observed.

[0053] A negative control experiment reveals that if cells are firstreacted with green mAb 3E7 in the presence of NHS and then washed andprobed with RTX, the two probes bind to the cells with completelydifferent patterns, and there is no evidence for co-localization.Moreover, the fluorescence pattern of binding of mAb 3E7 to the cells inNHS in the absence of RTX is weak. Similar patterns of complementopsonization and co-localization of mAb 3E7 with RTX were demonstrablein DB cells. In addition, the cross-linking described above was alsoobserved if only one or neither of the two key mAbs (RTX, 3E7) werelabeled with fluorescence dyes. These results provide further proof thatbinding of RTX to CD20 positive cells promotes robust complementactivation and C3b(i) deposition.

[0054] Finally, to further demonstrate that Rituximab and mAb1F5mediated cytotoxicity derives from complement activation, flow cytometrywas used to monitor the cytotoxic effects of various formulation (seeTables 2-5). Mab1F5 was previously used in a phase I trial for B celllymphoma, but it has not been commercialized and its mechanism of actionhas not been defined. Cells were incubated in the presence of media, orsera, ±Rituximab/3E7 etc. The readout is based on flow cytometry, theuptake of propidium iodide indicating cell death, and all experimentswere done in duplicate. Gating schemes are used to identify cells. G1are monodisperse cells, and G4 includes all cells. TABLE 2A 1 Hr 24 Hr48 Hr 72 Hr Raji (1 × 10⁵) G4 Live G4 Live G4 Live G4 Live Media 10,91620,328 61,755 79,637 10,075 19,791 59,396 77,878 Media + Ritux 9,8759,769 39,656 41,997 8,621 10,026 34,636 39,782 Media + 1F5 8,843 12,53940,717 46,051 9,741 14,218 47,101 53,389

[0055] TABLE 2B 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Total G4 TotalG4 Total G4 Total Media 12,694 23,258 65,119 85,085 11,791 22,608 62,72282,337 Media + Ritux 11,768 12,809 45,200 51,175 10,305 12,929 39,16347,065 Media + 1F5 10,437 14,973 44,596 52,591 11,502 17,111 51,16659,476

[0056] TABLE 2C 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G1 Live G1 Live G1Live G1 Live Media 10,596 20,010 60,328 44,109 9,747 19,467 58,05441,915 Media + Ritux 9,513 9,371 37,416 26,511 8,308 9,671 32,220 25,398Media + 1F5 8,541 12,377 39,065 25,428 9,328 14,011 45,364 30,440

[0057] TABLE 2D 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Dead G4 Dead G4Dead G4 Dead Media 1,036 1,810 2,120 2,746 979 1,727 2,033 2,240 Media +Ritux 1,081 2,085 2,986 4,309 956 1,977 2,469 3,285 Media + 1F5 9281,640 2,278 3,293 1,024 2,064 2,558 2,908

Tables 3A-D

[0058] TABLE 3A 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Live G4 Live G4Live G4 Live 50% Serum (O+) 6,201 14,783 24,956 29,536 5,844 13,12224,990 25,608 50% Serum (O+) 4,166 1,225 1,265 1,220 w/Rituximab 3,970915 884 1,529 50% Serum (O+) 549 695 3,632 3,677 w/1F5 626 611 5,0164,776 50% Serum (O+) 407 62 109 125 w/Rituximab+mAb 3E7 400 80 185 8550% Serum (O+) 41 58 312 259 w/1F5 + mAb 3E7 39 76 248 385

[0059] TABLE 3B 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Total G4 TotalG4 Total G4 Total 50% Serum (O+) 9,889 19,405 33,050 45,583 8,905 17,23632,043 40,910 50% Serum (O+) 9,126 12,508 10,690 11,059 w/Rituximab9,244 12,302 10,560 11,816 50% Serum (O+) 9,732 8,608 11,991 10,124w/1F5 9,470 8,388 15,102 12,852 50% Serum (O+) 5,082 5,561 6,695 7,991w/Rituximab+mAb 3E7 4,525 5,836 10,097 7,256 50% Serum (O+) 8,000 5,7787,247 6,834 w/1F5 + mAb 3E7 8,418 6,844 7,915 8,537

[0060] TABLE 3C 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G1 Live G1 Live G1Live G1 Live 50% Serum (O+) 5,979 14,287 21,807 14,597 5,630 12,60721,949 12,307 50% Serum (O+) 4,014 1,177 1,200 998 w/Rituximab 3,814 872823 1,229 50% Serum (O+) 490 677 3,495 3,141 w/1F5 545 611 4,826 4,15750% Serum (O+) 363 42 92 105 w/Rituximab+mAb 3E7 353 66 155 66 50% Serum(O+) 16 39 277 213 w/1F5 + mAb 3E7 14 65 219 344

[0061] TABLE 3D 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Dead G4 Dead G4Dead G4 Dead 50% Serum (O+) 1,763 2,522 3,583 3,847 1,641 2,304 2,9963,463 50% Serum (O+) 3,614 11,011 9,236 9,384 w/Rituximab 4,053 11,0959,529 9,734 50% Serum (O+) 8,680 7,503 7,483 5,494 w/1F5 8,334 7,4329,065 6,946 50% Serum (O+) 4,138 5,367 6,511 7,759 w/Rituximab+mAb 3E73,611 5,631 9,781 7,098 50% Serum (O+) 7,831 5,645 6,788 6,328 w/1F5 +mAb 3E7 8,225 6,689 7,915 7,789

Tables 4A-D

[0062] TABLE 4A 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Live G4 Live G4Live G4 Live 50% Serum (B+) 1,054 10,833 29,740 39,145 969 10,277 26,89336,869 50% Serum (B+) 3,972 1,366 530 797 w/Rituximab 4,500 1,120 941850 50% Serum (B+) 1,254 3,190 6,901 6,999 w/1F5 717 2,046 5,196 5,56150% Serum (B+) 183 63 114 132 w/Rituximab+mAb 3E7 143 48 70 77 50% Serum(B+) 38 89 706 417 w/1F5 + mAb 3E7 23 126 388 436

[0063] TABLE 4B 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Total G4 TotalG4 Total G4 Total 50% Serum (B+) 9,744 17,916 26,000 47,812 9,028 17,03124,000 45,643 50% Serum (B+) 9,182 11,800 10,064 10,481 w/Rituximab10,650 13,268 13,875 11,542 50% Serum (B+) 10,814 13,036 15,239 17,451w/1F5 11,310 12,674 14,599 13,698 50% Serum (B+) 3,667 3,930 6,895 5,608w/Rituximab + mAb 3E7 2,848 3,362 4,642 6,413 50% Serum (B+) 4,998 4,7759,565 7,765 w/1F5 + mAb3E7 6,739 5,968 7,804 8,269

[0064] TABLE 4C 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G1 Live G1 Live G1Live G1 Live 50% Serum (B+) 996 10,488 26,286 22,614 915 9,912 24,14321,380 50% Serum (B+) 3,746 1,283 499 652 w/Rituximab 4,290 1,058 873690 50% Serum (B+) 1,108 3,063 6,431 6,019 w/1F5 624 2,046 4,913 4,58050% Serum (B+) 149 44 108 103 w/Rituximab + mAb 3E7 118 35 48 62 50%Serum (B+) 16 74 641 357 w/1F5 + mAb3E7 9 104 353 371

[0065] TABLE 4D 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Dead G4 Dead G4Dead G4 Dead 50% Serum (B+) 4,743 5,586 5,615 5,627 4,501 5,274 5,6025,494 50% Serum (B+) 4,052 10,260 9,408 9,575 w/Rituximab 4,847 11,91812,799 10,572 50% Serum (B+) 9,023 9,500 8,002 9,986 w/1F5 10,281 10,3939,106 6,999 50% Serum (B+) 3,230 3,763 6,685 5,370 w/Rituximab + mAb 3E73,473 3,216 4,507 6,254 50% Serum (B+) 4,893 4,581 8,501 7,081 w/1F5 +mAb3E7 6,642 5,743 7,231 7,583

Tables 5A-D

[0066] TABLE 5A 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Live G4 Live G4Live G4 Live 50% Serum (A+) 7,097 14,796 30,167 33,584 6,246 13,50925,090 38,653 50% Serum (A+) 3,124 2,030 3,861 3,687 w/Rituximab 3,2211,669 2,054 3,329 50% Serum (A+) 1,646 2,446 10,484 9,167 w/1F5 1,0672,417 11,506 5,757 50% Serum (A+) 234 212 391 671 w/Rituximab + mAb 3E7380 213 341 1,420 50% Serum (A+) 28 121 198 999 w/1F5 + mAb3E7 26 99 3343,423

[0067] TABLE 5B 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Total G4 TotalG4 Total G4 Total 50% Serum (A+) 9,476 17,856 35,143 40,494 8,295 16,27529,255 45,669 50% Serum (A+) 8,918 12,737 18,557 18,218 w/Rituximab8,332 11,289 12,082 16,135 50% Serum (A+) 9,921 10,898 20,794 18,110w/1F5 9,717 10,696 21,634 14,075 50% Serum (A+) 3,967 7,102 8,866 10,045w/Rituximab + mAb 3E7 5,052 7,503 8,253 12,978 50% Serum (A+) 7,4956,062 6,162 8,277 w/1F5 + mAb3E7 6,299 7,057 4,859 13,438

[0068] TABLE 5C 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G1 Live G1 Live G1Live G1 Live 50% Serum (A+) 6,828 13,230 17,387 10,290 5,977 11,84816,161 13,339 50% Serum (A+) 2,856 1,864 2,973 2,685 w/Rituximab 2,9461,522 1,689 2,307 50% Serum (A+) 1,406 2,112 7,157 6,202 w/1F5 856 2,0887,937 3,895 50% Serum (A+) 204 188 323 540 w/Rituximab + mAb 3E7 348 195283 1,071 50% Serum (A+) 15 97 156 737 w/1F5 + mAb3E7 18 88 275 2,499

[0069] TABLE 5D 1 Hr 24 Hr 48 Hr 72 Hr Raji (1 × 10⁵) G4 Dead G4 Dead G4Dead G4 Dead 50% Serum (A+) 1,352 2,012 2,558 2,675 1,158 1,813 2,0802,809 50% Serum (A+) 4,131 10,128 12,929 13,292 w/Rituximab 3,690 9,0919,519 11,660 50% Serum (A+) 7,449 8,196 9,386 8,294 w/1F5 8,323 8,0478,831 6,330 50% Serum (A+) 3,393 6,563 8,199 8,796 w/Rituximab + mAb 3E74,197 6,947 7,658 10,254 50% Serum (A+) 7,379 5,810 5,850 6,696 w/1F5 +mAb3E7 6,177 6,866 4,349 8,681

[0070] In summary, Rituximab was found to effectively kill Raji cellsonly in the presence of serum. In almost all experiments, 3E7 (ananti-C3b(i) antibody) was observed to enhance the killing capacity ofRituximab. Similar results have been obtained with the mAb 1F5/mAb 3E7pair. See representative results in Tables 2-5. These results were allpart of a single experiment. Three different sera were used, as well asmedia.

[0071] Applicants contend that the most important mechanism of action ofRituximab requires complement activation, and thus CH50 values of thepatients' sera will be useful in assessing the effectiveness ofRituximab therapy. In principle the flow cytometric analysis paradigmcould be used to screen for the efficacy of Rituximab±3E7 in vitro,based on samples of blood taken from a patient, under conditions whichallow for complement activation. If the patient's CH50 is low, orkilling is weak, supplementation with fresh plasma from a compatibledonor or addition of one or more purified complement components would beindicated.

EXAMPLE 2

[0072] Administration of Rituximab Depletes Available Complement

[0073] Quantitative flow cytometry was used to measure RTX-mediatedC3b(i)-opsonization of ARH 77 cells (Tables 6A, 6B). In this procedurethe cells are reacted with NHS and RTX, and after a wash they are probedwith fluorescent labeled mAb 3C11, a mAb that is also specific forC3b(i). In parallel, calibrated fluorescent beads, which have knownnumbers of fluorescent molecules bound, are also examined by flowcytometry. In this way, by comparison to the bead standards, it ispossible to obtain a quantitative readout (MESF, molecules of equivalentsoluble fluorochrome) for the RTX-opsonized cells. The results indicatethat at higher cell concentrations the ability of serum to facilitaterobust C3b(i) opsonization in the presence of RTX decreases. A largeexcess of RTX was used in these experiments, and so a lack of RTX wasnot the reason for the decrease. Supplementation of the sera withpurified C2, either native, or oxidized (to further enhance Cactivation) clearly enhanced C3b(i) deposition, and this effect wasevident at lower serum concentrations as well.

[0074] As previously mentioned, it is likely that the limiting factor incomplement activity is C2, and this example shows that both C2 and itsmore stable oxidized form can enhance or restore C activity, as definedby opsonization. TABLE 6A Addition of Complement Component C2 EnhancesRituximab-mediated C3b(i) Opsonization* of ARH77 Cells in Serum MESHUnits Cell Concentration (cells/ml) 2 × 10⁶ 4 × 10⁶ 8 × 10⁶ Condition:50% serum + RTX 210,000 110,000 40,000 + RTX + C2 290,000 150,000100,000 25% serum + RTX 100,000 57,000 32,000 + RTX + C2 300,000 190,00067,000

[0075] TABLE 6B Addition of Complement Component C2 or Oxidized C2Enhances Rituximab-mediated C3b(i) Opsonization* of ARH77 Cells in SerumMESH Units Cell Concentration (cells/ml) 2 × 10⁶ 4 × 10⁶ Condition: 50%serum + RTX 360,000 240,000 + RTX + C2 420,000 290,000 + RTX + Ox-C2510,000 335,000 25% serum + RTX 270,000 160,000 + RTX + C2 380,000270,000 + RTX + Ox-C2 395,000 230,000

EXAMPLE 3

[0076] Supplementation of Patient Blood with Complement Enhances C3b(i)Deposition

[0077] Experimental evidence has been generated in support of theparadigm that supplementation with complement can enhance the efficacyof anti-cancer antibodies in killing their target cells. In particular,experiments on whole blood isolated from a patient with a B celllymphoma were performed and demonstrate that supplemental complement canenhance C3b(i) deposition. The whole blood was collected in citrate toprevent coagulation but the citrated plasma is still permissive forcomplement activation. Rituximab±an anti-C3b(i) (3E7) mAb was added tothe blood, and then flow cytometry and a phycoerythrin (PE)-labeled mAbspecific for C3b(i) (7C12, which recognizes an epitope different fromthat bound by 3E7) was used to measure deposition of C3b(i) on thecancer cells. The amount of C3b(i) deposited on the cancer cellsprovides a readout of the effectiveness of the Rituximab to activatecomplement in the medium (citrated plasma or serum) being tested. The3E7 mAb was use because it will enhance C3-mediated deposition promotedby Rituximab. The entire experiment was conducted in a parallel study inwhich the patient's blood cells were washed several times and thenreconstituted in an AB+ plasma from a normal healthy blood donor.Control experiments were conducted in blood anti-coagulated with EDTA,because complement activation is completely blocked by EDTA.

[0078] The results shown in Table 7 clearly indicate that complementactivation in the patient's own plasma is quite modest, as manifested inthe weak staining of the cancer cells with the PE-labeled anti-C3b(i)mAb, 7C12. However, when plasma from a healthy normal donor wassubstituted for patient plasma, a very high level of C3b(i) depositionwas demonstrable on the cancer cells. Thus, supplementation ofcomplement by iv infusion of normal compatible donor plasma is clearlyindicated for this patient. TABLE 7 PE-7C12 staining Log Mean PlasmaAdditions Fluorescence Citrated patient Nothing <15 Rituximab 180Rituximab and mAb 3E7 215 Citrated normal (AB+) Nothing <60 Rituximab1723 Rituximab and mAb 3E7 1821 EDTA patient Nothing <15 Rituximab <50Rituximab and mAb 3E7 <50

[0079] The magnitude of the log mean fluorescence is indicative of thedegree of C3b(i) opsonization induced by Rituximab. In EDTA plasma,where complement is blocked, or in the patient's citrated plasma, C3b(i)deposition is quite low. Rituximab was added at 10 ug/ml and mAb 3E7 wasadded at 3 ug/ml.

EXAMPLE 4

[0080] RTX-Mediated Killing of DB Cells by Complement is Enhanced byAddition of Complement Component C2.

[0081]FIGS. 1A and 1B illustrate the results of an experiment in whichflow cytometry was used to measure RTX-mediated killing of DB cells overthe course of a 24 hour incubation. The general methodology wasidentical to that previously reported in Tables 2-5 for Raji cells. Atthe lower cell concentrations RTX-mediated killing is moderately good,especially at a RTX concentration of 100 ug/ml (see FIG. 1A). However,at higher cell concentrations (3.6×10⁶ cells/ml vs 4.0×10⁵ cells/ml),even the use of 100 ug/ml RTX leaves more than half of the cells aliveafter 24 hours (see FIG. 1B). It is likely that the limiting factor inthis killing assay is the capacity of complement to kill so many cells.In fact, supplementation of the serum with C2 leads to a substantialincrease in killing, as illustrated in the large drop in live cells inthe presence of both RIX and C2. Note that C2 by itself in serum doesnot promote killing. This example provides additional evidence thataddition of C2 can enhance killing of cancer cells, even when thecomplement source is from a normal donor.

EXAMPLE 5

[0082] Binding of RTX to Large Numbers of Cells Depletes Complement, andAddition of C2 can Restore Complement Activity.

[0083] In this experiment RTX (10 or 100 ug/ml) was incubated withtarget cancer cells in NHS for one hour at 37 degrees C., and then thecells were pelleted and the residual level of complement in the NHS wasmeasured in a CH50 assay. Although there was no drop in complementactivity if the cell concentrations were 10⁶ per ml or lower, at highercell concentrations consumption of complement was clearly evident. Infact, at cell concentrations of 108 per ml, more than 80% of thecomplement was consumed due to binding of RTX to the cells and formationof complement-fixing immune complexes. There was no loss of complementactivity for cells alone or for RTX alone.

[0084] The results illustrated in Table 8 indicate that the major reasonfor the loss of complement activity must have been due to consumption ofcomplement component C2. That is, when C2 is added to the depletedsupernatants (SN), in all but one case full complement activity isrestored. This study is relevant because in certain hematologicmalignancies the burden of tumor cells in the circulation can reach 10⁸per ml. Alternatively, binding of anti-tumor mAbs to tumor masses inorgans, of ˜200 ml or more would also be expected to substantiallydeplete complement. This experiment argues that C2 may be sufficient inmany cases to restore complement activity and restore the killingcapacity of the anti-tumor mAb. TABLE 8 Restoration of ComplementActivity by Addition of Complement Component C2 CH50 SN from 1 hr 37° C.10 ug/ml RTX 100 ug/ml RTX incubation with No Addition of No Addition 1× 10⁸ cells/ml addition C2 addition of C2 ARH-77 108 >300 57 252 Raji127 >300 46 297 DB 84 266 28 117

[0085] Cells+serum+RTX were incubated for 1 hour, resulting inconsumption of complement. After the cells were pelleted, thesupernatants were tested for complement activity (±C2) in a CH50 assay.Naive serum had a CH50 of ˜275 (normal range,150-300).

EXAMPLE 6

[0086] Treatment of a Chronic Lymphocytic Leukemia Patient withRituximab Leads to Substantial and Prolonged Depletion of Complement.

[0087] The complement activity of a Chronic Lymphocytic Leukemia (CLL)patient receiving RTX treatment was monitored over the course of thefour weeks of treatment. The results illustrated in Table 9 indicatethat although the patient had a high level of complement activity beforeRTX treatment, his complement levels were severely reduced as aconsequence of treatment. The assay results are based on a completemulti-point titration (serum/5 to serum/320) for each serum sample, andare normalized to 100 for the initial serum sample (pre-treatment). Inthis assay, each serum dilution is tested, in duplicate, for its abilityto facilitate lysis of antibody-sensitized sheep red blood cells.Selected samples were also tested for CH50 at the UVA clinical lab,which uses a single serum concentration to lyse sensitized sheep redblood cells; this later assay is therefore based in part onextrapolation. The CH50 value for the pre-treatment bleed was 242. Theresults for the next 8 consecutive bleeds are: 242, 68, 204, 167, 89,60, 56, 58. Thus, the results obtained by the clinical lab also indicatea profound reduction of complement activity, but some of the singlepoints tend to be higher than those obtained by the more completemultipoint assay. These findings also suggest that more careful andcomprehensive analyses of complement activity, rather than a singlepoint clinical determination, will be required to fully assess thecomplement status of a patient. Such assays would include the completemultipoint assay as well as tests with RTX and cancer cells, asmentioned above. TABLE 9 Decrease in Complement Activity During theCourse of RTX Treatment Week 1 Week 2 Week 3 Week 4 Pre-infusion 100 50≦10 ≦10 1 hour 90 30 <10 ≦10 Post-infusion 20 30 <10 ≦10

[0088] Each week the CLL patient received 375 mg/m² of RTX, IV over 6-8hours. The pre-infusion sample was taken before the start of eachinfusion. The 1 hour sample was taken after 10-15% of the infusion wascomplete, and the post-infusion sample was taken soon after the infusionwas completed. Values are normalized to 100 for the initial pre-infusionsample.

1. A method of enhancing the complement mediated cytotoxic activity ofan anti-neoplastic antibody for its target cells, said method comprisingthe steps of contacting said cells with the anti-neoplastic antibody;and contacting the cells with a composition comprising a donorcomplement protein.
 2. The method of claim 1, wherein the antibody is amonoclonal antibody.
 3. The method of claim 1, wherein the antibody isselected from the group consisting of Rituximab and Campath.
 4. Themethod of claim 1, wherein the donor complement composition comprisesserum or plasma.
 5. The method of claim 1, wherein the donor complementcomposition comprises a purified member of the complement cascade. 6.The method of claim 5, wherein the purified member is a recombinantprotein.
 7. The method of claim 5, wherein the purified member comprisesa protein factor selected from the group consisting of C2, C3, C4,factor B, factor D and properdin.
 8. The method of claim 5, wherein thepurified member is C2.
 9. The method of claim 8, wherein the C2 isoxidized.
 10. A kit for enhancing the cytotoxic activity of anti-tumorantibodies, said kit comprising a composition wherein the compositioncomprises a complement protein.
 11. The kit of claim 10 furthercomprising an anti-cancer antibody.
 12. The kit of claim 11, wherein theantibody is selected from the group consisting of Rituximab and Campath.13. The kit of claim 10, wherein the complement composition comprisessera or plasma.
 14. The kit of claim 10, wherein the complementcomposition comprises a purified member of the complement cascade. 15.The kit of claim 14, wherein the purified member is a recombinantprotein.
 16. The kit of claim 14, wherein the purified member comprisesa protein selected from the group consisting of C2, C3 and C4.
 17. Thekit of claim 16, wherein the purified member is C2.
 18. A compositionfor treating patients with a neoplastic disease, said compositioncomprising an anti-neoplastic antibody; and a complement protein. 19.The composition of claim 18 wherein the composition comprises serum orplasma isolated from AB type blood.
 20. The composition of claim 18wherein the composition comprises purified C2 protein.
 21. A method foridentifying individuals that would benefit from supplementing ananti-cancer antibody therapy with donor complement, said methodcomprising the steps of: providing a serum or plasma sample from theindividual to be treated; contacting the sample with cancer cells andthe anti-cancer antibody to form a reaction mixture; incubating thereaction mixture in the presence and absence of donor complementproteins; and determining whether the sample exhibits higheropsonization or cell killing in the presence of supplement complement asthe criteria for identifying a individual that would benefit fromcomplement supplementation.
 22. The method of claim 21 wherein thecancer cells are isolated from the individual.
 23. The method of claim21 wherein the cancer cells are an established cell line that serves asa model for the cancer type afflicting the individual.
 24. The method ofclaim 21 wherein the donor complement protein is provided as matchedserum or plasma from a healthy individual.
 25. The method of claim 21,wherein the donor complement protein comprises a purified member of thecomplement cascade.
 26. The method of claim 21, wherein the donorcomplement protein is C2.